Effect of degumming degree on the structure and tensile properties of RSF/RSS composite films prepared by one-step extraction

Regenerated silk fibroin (RSF) and regenerated sericin (RSS) have attracted much attention for tissue engineering due to excellent biocompatibility and controllable degradation. However, pure RSF films prepared by existing methods are brittle, which limits applications in the field of high-strength and/or flexible tissues (e.g. cornea, periosteum and dura). A series of RSF/RSS composite films were developed from solutions prepared by dissolving silks with different degumming rates. The molecular conformation, crystalline structure and tensile properties of the films and the effect of sericin content on the structure and properties were investigated. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction results revealed more β-sheets in films prepared by boiling water degumming than in Na2CO3-degummed RSFC film. Analysis of mechanical properties showed that the breaking strength (3.56 MPa) and elongation (50.51%) of boiling water-degummed RSF/RSS film were significantly increased compared with RSFC film (2.60 MPa and 32.31%), and the flexibility of films could be further improved by appropriately reducing the degumming rate.

www.nature.com/scientificreports/ Sericin is a globular protein with high viscosity that can protect core fibres and is rich in hydrophilic groups 14 . In general, sericin is usually discarded as waste in the preparation of RSF materials, leading to environmental pollution 15 . Regenerated sericin (RSS) also possesses good biocompatibility, but its use in biomaterials has not been explored due to concerns over immunogenicity, but this was recently addressed 16 . Accordingly, RSS has been explored for use in wound dressing, cornea repair, bone/cartilage repair and drug delivery [17][18][19][20] . The molecular weight of sericin also varies depending on the extraction method. The molecular weight of RSS collected by Na 2 CO 3 degumming was significantly lower than that obtained by boiling water degumming 21,22 . In addition, a study indicated that silk fibroin fibres can be destroyed by the complete removal of sericin during degumming 23 , inevitably reducing the molecular weight of RSF.
We developed a boiling water degumming method to obtain RSF/RSS composite solutions by controlling the degumming rates in order to retain high-molecular-weight silk protein 24 . This method was not necessary to prepare RSF and RSS separately and then blend them, hence we considered it a one-step extraction method. Subsequently, a series of RSF/RSS composite films were prepared by polyethylene glycol diglycidyl ether (PEG-DE) crosslinking 25 and the structures and tensile properties of the films were investigated to explore their application prospects for tissue engineering. The present work provides a new method to obtain silk protein films with improved flexibility using fewer chemicals and without loss of sericin, benefitting environmental protection.

Materials and methods
Materials. Bombyx  Degumming and dissolving of silk. B. mori raw silk was treated in boiling distilled water at a bath ratio of 1:50 (w/v) for 1, 3 or 5 h, and boiling distilled water was replaced every hour. Na 2 CO 3 (0.06 wt%) degumming served as a control 26 . After drying, silks with various degree of degumming (Table 1) were dissolved in 9.3 M LiBr at 65 °C for 1 h, then dialysed against distilled water at 4 °C for 72 h. Finally, silk protein aqueous solutions were concentrated to 50 mg/mL.

Preparation of PEG-DE-crosslinked films. PEG-DE was added to silk protein aqueous solution drop-
wise with stirring at a weight ratio of 1.0:0.8, then debubbled. All films were prepared by casting the same volume of mixture into polyethylene dishes (diameter 50 mm), then drying at 40 °C for 6 h while revolving slowly. Noncrosslinked films served as controls.
Measurement of mechanical properties. The tensile properties of degummed silk fibres and films were measured using an Instron 3365 universal testing machine (Instron, Boston, MA, USA) at 20 ± 2 °C and 65 ± 2% relative humidity (RH). Degummed silk fibres were preconditioned under the above temperature and humidity conditions for 24 h before measurement. Parameters were as follows: clamp distance 250 mm, extension rate 250 mm/min, pre-tension 0.5 cN. The linear density (tex) of fibres was determined by measuring the length and mass of filaments (Eq. 1), and the specific breaking strength (N/tex) was calculated according to Eq. 2.
All films were cut into rectangles of 10 mm × 50 mm, immersed in deionised water for 1 h, and the thickness was measured by an electronic spiral micrometre. The tensile properties of films were measured in the wet state. Parameters were as follows: clamp distance 20 mm, extension rate 20 mm/min, pre-tension 0.5 cN. For each fibre or film, 10 independent samples were tested. Samples were secured into the Instron clamps and tests were run until samples failed through tearing. Breaking strength (MPa) and the breaking elongation (%) were calculated according to Eqs. (3) and (4) respectively: www.nature.com/scientificreports/ where Nt (tex) is the linear density of fibres, G k (g) is the quality of fibres with conventional moisture regains, F (N) is the breaking force, S (mm 2 ) is the cross-sectional area of films, L (mm) is the breaking length of samples, and L 0 (mm) is the original length of the samples. Young's modulus (MPa) was calculated using the secant moduli from 5 to 10% strain in the stress-strain curves.
Structure characterisation. The chemical structure and molecular conformation of all films were analysed using a Nicolet Avatar-IR360 Fourier transform infrared spectroscopy (FTIR) instrument (Nicolet, Madison, WI, USA). Thirty-two scans were recorded with a resolution of 4 cm −1 and a scanning range of 500-4000 cm −1 .
In addition, we semi-quantitatively analysed the molecular conformation by deconvolution of the amide I band using Peakfit v4.12 software as reported previously 24 .
Crystalline structures of all samples were determined by a X 'Pert-Pro MRD X-ray diffractometer (XRD; Philips, Amsterdam, The Netherlands) with a CuK α radiation source at 2θ of 5-50° and a scanning speed of 2°/min. Crystallinity was calculated through separate peak-fitting using the same quantitative analysis software as for FTIR.

Statistical analysis.
Results are presented as mean ± standard deviation (SD) of the mean. Comparison of means was performed using one-way analysis of variance (ANOVA), followed by independent Student's t-tests using SPSS17.0 statistical software (IBM, Armonk, NY, USA). Statistical significance was set at p < 0.05.

Results and discussion
Tensile properties of degummed silk fibres. The surface of raw silk covers a complete layer of sericin that binds two fibroin fibres tightly together 27 . Sericin can be dissolved in hot water. The degumming method or degumming degree can have a significant impact on the mechanical properties of silk fibres. Therefore, we measured the mechanical properties to evaluate the degree of damage for degummed silk fibres with different degumming rates. The degumming rate of raw silk was stable after degumming in boiling water for 5 h, which was close to that of Na 2 CO 3 degumming. As shown in Fig. 1, the specific breaking strength and breaking elongation of raw silk decreased after degumming, especially for Na 2 CO 3 -degummed silk fibres, which decreased by 26% and 38%, respectively. Removing sericin weakened the cohesion between single fibres, hence the tensile properties of silks were significantly decreased. However, the specific breaking strength and the breaking elongation of SF fibres were significantly higher than those of SF C fibres, indicating that Na 2 CO 3 degumming caused serious damage to silk fibroin macromolecules. Due to the discontinuous distribution of residual sericin, many weak regions were generated on the surfaces of silks, which resulted in lower specific breaking strength and breaking elongation for SF/SS 1 and SF/SS 2 fibres compared with SF fibres.
In our preliminary experiments, repeated tests demonstrated that the Na 2 CO 3 degumming rate was slightly higher than that of boiling water degumming for 9 h, indicating that the hierarchical structure or macromolecular chains of silk fibres were destroyed by Na 2 CO 3 degumming, resulting in partial dissolution of silk fibroin 28 . in a wet state, hence the effect of the degumming degree on the tensile properties of wet films was examined. Figure 2a shows the stress-strain curves of PEG-DE-crosslinked films. The mechanical properties of regenerated silk protein-based materials depend on their molecular weights and crystallinities; the higher the molecular weight, the stronger the mechanical properties 29 . Therefore, compared with RSF C film prepared by Na 2 CO 3 degumming, the breaking strength of RSF films was increased significantly. This result is consistent with those obtained by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in our previous study; thus, boiling water degumming causes less damage to macromolecules of fibroin fibres, resulting in higher molecular weight compared with Na 2 CO 3 degumming 24 . In the presence of sericin, the breaking elongation of RSF/RSS composite films was significantly improved, and dependent on the sericin content (Fig. 2b). Sericin macromolecules shuttled between silk fibroin macromolecular chains, and bound to silk fibroin macromolecules via hydrogen bonds or covalent bonds. Therefore, when the films were stretched, silk fibroin macromolecular chains sharply extended due to the "bridging effect" and relative slippage of the sericin macromolecules 30 . In addition, a small amount of sericin enhanced the force between silk fibroin macromolecular chains, resulting in increased breaking strength of RSF/RSS 2 composite films. Figure 2c shows that the breaking strengths were ordered RSF/RSS 2 film (3.56 ± 0.37 MPa) > RSF film (3.24 ± 0.39 MPa) > RSF C film (2.60 ± 0.13 MPa). The breaking strengths of RSF/RSS 2 and RSF films were equivalent to natural cornea tissue (approximately 3-5 MPa) 31 , and higher than that of RSF-based biomimetic periosteum prepared by Na 2 CO 3 degumming 32 . Therefore, RSF film and RSF/RSS 2 composite film were considered alternative hard-tissue repair materials such as dura or periosteum. However, when the degumming rate was lower, the higher content of sericin resulted in an increase in solution viscosity and disruption to the ordered arrangement of silk fibroin macromolecular chains 24 , leading to reduced breaking strength of RSF/RSS 1 film to even less than that of RSF C film prepared by Na 2 CO 3 degumming.
Compared with RSF C film, the Young's modulus of RSF film was increased, while that of RSF/RSS 1 film and RSF/RSS 2 film was decreased significantly (Fig. 2d). Notably, breaking elongation of RSF/RSS 2 film (50.51%) was 1.6 times that of RSF C film, and was significantly higher than the elongation of a propionamide/SF blend film (~ 13%) or poly(ε-caprolactone)/SF electrospun film (38.14%) used in corneal regeneration 33,34 . These results indicated that the flexibility of RSF/RSS 2 film was markably improved, making it a candidate material for corneal tissue repair. www.nature.com/scientificreports/ Molecular conformation of PEG-DE-crosslinked films. For silk protein-based materials, the secondary structure and crystalline structure formed by self-assembly are key factors determining the mechanical properties 35 . Figure 3 shows the FTIR spectral curves of PEG-DE crosslinked films. All films showed similar characteristic bands at ~ 1618 cm −1 (β-sheet), 1515 cm −1 (β-sheet) and 1233 cm −1 (β-sheet), which were assigned to the C=O stretching vibration, N-H bending vibration and C-N stretching vibration, respectively 36 . Compared with non-crosslinked films 24 , the amide I peak of crosslinked films moved from 1637 cm −1 to 1618 cm −1 , indicating a change from random coil to β-sheet conformation. Under the polarity effect of sericin macromolecules, PEG-DE further induced the silk fibroin macromolecular chains to extend in an orderly manner, which promoted more amide groups to form hydrogen bonds and weakened the stretching vibration of C=O. In addition, the characteristic peak near 3276 cm −1 became broader and stronger, indicating that more -OH groups formed new hydrogen bonds between macromolecules. Compared with RSF C film, the stronger intensity at 3276 cm −1 for RSF/RSS composite films was ascribed to the stronger association action of -OH 37 . Table 2 shows the molecular conformational contents of all films. In non-crosslinked films, the β-sheet content of RSF film was significantly higher than that of RSF C film. This result further confirmed that boiling water degumming produced high-molecular-weight silk protein, which contributed to the formation of more stable β-sheet structure. The β-sheet content of PEG-DE-crosslinked films was obviously increased, especially in boiling water degumming groups. PEG-DE can chemically react with OH, -COOH and NH 2 groups in silk fibroin and sericin macromolecules, which promotes crosslinking of silk protein macromolecules to form stable β-sheet structure 38 . A small amount of sericin slightly increased the β-sheet content of RSF/RSS 2 composite film owing to an increase in functional groups. In contrast, excessive sericin in RSF/RSS 1 composite film inhibited the orderly self-assembly of silk fibroin, resulting in relatively less β-sheet content. Thus, the β-sheet content was ordered RSF/RSS 2 composite film > RSF film > RSF/RSS 1 composite film, consistent with the breaking strengths of these films.

Crystalline structures of PEG-DE-crosslinked films.
The H-chain is the major protein component with a regular structural sequence and accounts for ~ 92% of the molecular weight of silk fibroin. The H-chain  www.nature.com/scientificreports/ is composed of 12 hydrophobic regions and 11 hydrophilic regions arranged alternately 39 . The highly repetitive (AGSGAG)n sequences induce silk fibroin macromolecules to aggregate and form a dense crystalline structure 40 . Sericin, which surrounds two strands of fibroin fibres in raw silk, is an amorphous material and soluble in hot water 41 . However, RSS material can form partially crystalline aggregates induced by the hydrogen bonding effects of hydrophilic groups during the process of macromolecular self-assembly. In our previous study, there were obvious silk I crystalline diffraction peaks at 12.1° and 19.9° for non-crosslinked RSF/RSS and RSF films, and 24.7° for RSF C film 24 , indicating that silk I crystals in RSF/RSS or RSF films were more abundant than in RSF C film. After PEG-DE crosslinking, typical silk II crystalline peaks appeared at 9.1°, 20.7° and 24.3° for all films (Fig. 4), which indicated that PEG-DE converted silk I into silk II (β-sheets). Furthermore, the silk II crystalline peak of sericin appeared at 23.3°4 2 , which enhanced the crystalline peak at 24.3° for silk fibroin, especially for RSF/RSS 1 film. The results also confirmed that the higher the molecular weight, the more stable the aggregation structure formed under the induction of external factors.

Conclusion
In this study, a one-step extraction method was used to prepare RSF/RSS composite solutions with uniform distribution. We developed a series of PEG-DE-crosslinked RSF/RSS composite films and RSF films with improved mechanical properties compared to those of RSF C film degummed by Na 2 CO 3 . Analysis of tensile properties indicated that boiling water degumming caused less damage to fibroin fibres. Further studies showed that PEG-DE-crosslinked films prepared using boiling water degumming formed stable secondary structures and crystalline structures, and the β-sheet content was significantly higher than that of RSF C film prepared by Na 2 CO 3 degumming. Films prepared using boiling water degumming possessed significantly enhanced breaking strength and flexibility. The study provided new strategies for application of silk protein in dura, periosteum and corneal tissue repair. In addition, the full utilisation of sericin and the absence of chemicals in the degumming process were of benefit to environmental protection.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. www.nature.com/scientificreports/